Thymidine kinase 1, TK1, (ATP: thymidine 5′-phosphotransferase, EC 2.7.1.21) is an enzyme involved in DNA precursor synthesis. A form of TK1 is present at high levels also in sera from humans and animals with malignant tumors. Therefore, serum TK1 activity measurements have been used for monitoring and for prognostic purpose in several different malignant diseases, but primarily in case of leukemia and lymphoma.
Furthermore, TK1 is the only proliferation marker that can be determined in the blood and it is likely to provide a large clinical benefit if available as a routine laboratory test [1-8].
The serum TK1 activity has been measured using a radioactive substrate 125I-dUrd (the PROLIFIGEN® TK-REA, DiaSorin Inc) for several decades, but this radio-enzymatic assay has had limited use and only in case of malignant hematologic malignancies [1, 2, 6]. A non-radiometric TK1 activity assay (TK LIAISON® assay, DiaSorin Inc.) has been available in recent years. This is a sensitive and robust assay and has provided clinically valuable information in humans and dogs with hematologic malignancies, particularly for monitoring therapy and predicting relapse [8, 9].
During the last 15 years antibodies against human TK1 have been available and enabled the determination of TK1 protein levels, in contrast to TK1 activity, in different hematologic and solid tumors, such as breast carcinomas [5, 10, 11], and several other forms of solid and hematologic tumors [11, 12].
The TK1 protein determinations rely mainly on a dot blot procedure based on anti-TK1 antibodies produced against the C-terminal part of TK1 [3, 5, 10-13]. The main reason for choosing this strategy for antibody production is that the C-terminal region is involved in the cell cycle regulation of TK1 [14-16]. It contains a recognition sequence for initiating the degradation of TK1 during mitosis and it has been assumed that this is an exposed region to which it could be possible to generate antibodies [3, 8, 13, 20, 23-25]. Although the dot blot assay has been used successfully in a number of studies [5, 10-13] the major limitation is that it is not a routine method in clinical laboratory practice. There are now more than six different anti-TK1 antibody preparations available commercially and several have been shown to react with recombinant and cellular TK1 (www.acris-antibodies.com). However, there is at present no antibody based assay for serum TK1 in routine clinical practice.
The main reason for the difficulty in establishing a sensitive and robust assay for serum TK1 is most likely related to the complex and variable forms of TK1 present in blood. Recently, several studies, both with the human and dog TK1 enzymes, have been conducted characterizing the recombinant, cellular and serum forms of TK1 [17]. It was found that TK1 exists in multiple oligomeric complexes and importantly only a sub-fraction of these complexes were enzymatically active. This is particularly the case for serum TK1 in patients with solid tumor diseases. The oligomerization seems to be related to the formation of disulfide cross linking occurring in the blood, but most likely also when the tumor cells disintegrate and TK1 is transported into the blood stream. Treatment with reducing agents leads to some reorganization of the serum TK1 complexes [2, 17], but the consequences of these facts for immune assay design have not so far been addressed. The fact that a major part of the TK1 protein in blood is enzymatically inactive may explain that TK1 enzyme activity measurements are not very effective in case of solid tumor diseases [18]. Thus, there is a large need to establish routine in vitro diagnostic procedures that can measure serum TK1 with sufficient sensitivity for clinical use.